By the end of July 2021 Zimbabwe, has reported over 100,000 SARS-CoV-2 infections. The true number of SARS-CoV-2 infections is likely to be much higher. We conducted a seroprevalence survey to estimate the prevalence of past SARS-CoV-2 in three high-density communities in Harare, Zimbabwe before and after the second wave of SARS-CoV-2.Between November 2020 and April 2021 we conducted a cross-sectional study of randomly selected households in three high-density communities  in Harare. Consenting participants answered a questionnaire and a dried blood spot sample was taken. Samples were tested for anti-SARS-CoV-2 nucleocapsid antibodies using the Roche e801 platform.A total of 2340 individuals participated in the study. SARS-CoV-2 antibody results were available for 70·1% (620/885) and 73·1% (1530/2093) of eligible participants in 2020 and 2021. The median age was 22 (IQR 10-37) years and 978 (45·5%) were men. SARS-CoV-2 seroprevalence was 19·0% (95% CI 15·1-23·5%) in 2020 and 53·0% (95% CI 49·6-56·4) in 2021. The prevalence ratio was 2·47 (95% CI 1·94-3·15) comparing 2020 with 2021 after adjusting for age, sex, and community. Almost half of all participants who tested positive reported no symptoms in the preceding six months.Following the second wave, one in two people had been infected with SARS-CoV-2 suggesting high levels of community transmission. Our results suggest that 184,800  SARS-CoV-2 infections occurred in these three communities alone, greatly exceeding the reported number of cases for the whole city. Further seroprevalence surveys are needed to understand transmission during the current third wave despite high prevalence of past infections.GCRF, Government of Canada, Wellcome Trust, Bavarian State Ministry of Sciences, Research, and the Arts Many national and subnational population-based SARS-CoV-2 seroprevalence surveys have been conducted globally and summarised in a recently published systematic review. Pooled estimates of SARS-CoV-2 seroprevalence in the general population varied greatly by WHO region with 19·6% , 6·8% , 4·7%  testing positive for SARS-CoV-2 antibodies in the South East Asian, American and European regions respectively. Overall the pooled estimated ratio of serologically detected infections to confirmed cases of COVID-19 was 11·1 (95%CI 8·3-14·9) suggesting that for each virologically confirmed SARS-CoV-2 infection, at least ten infections remained undetected by surveillance systems globally. Only two population-based studies from Africa contributed to the systematic review reporting a SARS-CoV-2 prevalence of 8·8% in Ethiopia in April 2020 and 25·4% in Nigeria in August 2020. Both studies had relatively small sample sizes and used rapid antibody tests. We searched PubMed up to June 19, 2021, for peer-reviewed and preprints using the search terms “COVID-19” OR “SARS-CoV-2” AND “prevalence” AND “Africa”. Additionally, we searched bibliographies of identified studies, a database of seroprevalence studies maintained by WHO, and the Google search engine for manuscripts and non-peer reviewed pre-prints. We identified three more studies conducted in Africa reporting SARS-CoV-2 seroprevalence estimates for the general population. Prevalence was 2·1% in Zambia in July 2020, 38·5% in South Sudan in August-September 2020 and 26% in rural and 41% in urban South Africa in March 2021. The South African study was a cohort study and estimated the prevalence following the first and second SARS-CoV-2 wave.To our knowledge, this is the first population-based SARS-CoV-2 prevalence study done in a low-income country in sub-Saharan Africa, to estimate cumulative SARS-CoV-2 prevalence after the second wave. Despite lower than predicted hospitalisations and deaths due to COVID-19 in Zimbabwe, SARS-CoV-2 seroprevalence in the population is extremely high across age-groups. Seroprevalence more than doubled during the second wave.This study demonstrates that laboratory-confirmed case notifications grossly underestimate the true number of infections in Zimbabwe. The majority of SARS-CoV-2 infections were asymptomatic. These findings are crucially important for future SARS-CoV-2 control measures and more specifically for vaccination strategies. If the majority of the population has some immunity due to past infection, a single-dose vaccination approach may be an option. Longitudinal studies in low-income settings in rural and urban populations are urgently needed to understand optimal vaccination strategies in the context of prior infection.Alt-text: Unlabelled box1The World Health Organization (WHO) declared the SARS-CoV-2 outbreak a global pandemic on March 11 2020, following the identification of a cluster of cases of pneumonia, later termed COVID-19, caused by a novel coronavirus (SARS-CoV-2), in Wuhan China in December 2019 A nationwide lockdown was first implemented on March 30 2020, confining people to their homes, and restricting movement to a 5 km radius. All air and land borders were closed to foreign nationals, only allowing returning residents and nationals to enter the country. Use of face coverings and social distancing was mandated, and all public transport, apart from essential state worker transport , was suspended. The lockdown was extended until May 17 2020 By July 27 2021 Zimbabwe has reported a total of 101,711 confirmed SARS-CoV-2 infections and 3280 deaths  [6]. MosLittle information is available about the prevalence of SARS-CoV-2 in the general population in Africa Differences in population demographics, living conditions, in-country mobility, risk of virus importation, disease epidemiology, health system access and climate may affect SARS-CoV-2 epidemiology. Large representative population-based seroprevalence surveys are needed to understand the epidemiology of SARS-CoV-2 in Africa and estimate infection rates, calculate infection-hospitalisation and infection-fatality ratios and compare infection burden between waves. These data are crucial to guide public health responses including vaccination strategies.We report a population-based household SARS-CoV-2 seroprevalence survey conducted in three high-density communities in Harare, Zimbabwe between November 2020 and April 2021. We also compared seroprevalence before (November – December 2020) and after (February – April 2021) the second wave.22.1We conducted a cross-sectional study on randomly selected households aiming for a total of 2000 participants in three communities in Harare namely Budiriro, Highfield and Mbare, the largest and most densely populated communities in the city.A sample size of 2000, assuming a design effect of 2 for the variance inflation due to clustering at household level and community level, was calculated to provide adequate precision around a prevalence of 5% . Antibody prevalence was not expected to be lower than 5% in the population.Fieldwork started on November 20 2020 and ended on April 17 2021 with a five-week hiatus due to a nationwide lockdown in January 2021 . In Budiwww.maps.me) on the researcher's tablet. Upon arrival the plot closest to the GPS point was selected for the survey. If on a plot there were multiple households living in a house, or multiple houses or flats all households were numbered, and a random number generator (Random Number Generator Plus by Random Apps Inc.) was used to randomly select one household.Households were selected through randomly generated GPS coordinates using the QGIS software (version 3.12). All GPS points were loaded onto the application ‘Maps.Me’ for Android , information about any visitors staying in the household overnight and whether or not the visitor had recently travelled to Zimbabwe from outside the country was also collected.https://www.surveycto.com/). Dried blood spot (DBS) samples were obtained from all consenting participants using finger prick technique onto a filter card. If household members were not present at the initial visit, then the research team arranged two further follow up visits to enrol all members of the household. If after three visits a household member was still not present, they were not included.For each participant, an individual-level questionnaire was administered that included information about demographics, occupational status, medical history, SARS-CoV-2 exposures, and history of any symptoms of viral diseases over the past 6 months. From February 2021 onwards questions about travel and sleepovers in other households, as well as attendance at large events (>50 people) were added. All questionnaires were administered using electronic data capture software . Prevalence was calculated as number of positive tests divided by number of samples tested, per district and by year (representing before and after the second wave). Age was categorised in 10-year bands for those aged 10+ years and 5-year bands for those under 10. Non-response weights were calculated using age band and sex from the sampling frame of all eligible individuals at participating households, including those who refused to participate or were absent. Separately we calculated prevalence by year adjusting for sensitivity and specificity of the assay 2.5The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. AF, VS, TB, NR, RAF and KK had access to the data. The decision to submit the manuscript for publication was a joined decision taken by all authors.3Between November 20 and December 20, 2020 , we apprIn both years, the study included more children and young adults than older individuals . The medIn 2020, 19·0% (95% CI 15·1-23·5%) of participants were SARS-CoV-2 positive and in February-April 2021 the SARS-CoV-2 prevalence was 53·0% (95% CI 49·6-56·4). The prevalence ratio was 2·48 (95% CI 1·94-3·16) comparing 2020 with 2021 after adjusting for age, sex and community . Those l4According to our data one in two people  had been infected with SARS-CoV-2 by April 2021 in three high density communities in Harare, Zimbabwe. The seroprevalence survey results suggest that 184,800 172,900-196,700) SARS-CoV-2 infections occurred in these three communities alone, which greatly exceeds the reported number of cases for the whole city . Unfortunately, notified SARS-CoV-2 infections are only reported by province and notifications by district or subdistrict are not available. Hence direct comparisons between the number of infections estimated from the seroprevalence survey and notification data are difficult. The three communities have an estimated population of 350,000, which accounts for 19% of the population in the capital [6,700 SAROur data confirm that there has been extensive community transmission during the first and second wave. The data from two communities in Harare showed that 18·0% 95% CI 14·1-22·1%) of the population had been infected with SARS-CoV-2 following the first wave. The prevalence of past infection was highest among the 30–39 year age-group with no difference between men and women, which is comparable to other data from sub-Saharan Africa ·1-22·1% ,21. SARSFollowing the second wave, SARS-CoV-2 seroprevalence was higher in the population of Mbare compared to the prevalence in the other two communities. Mbare is the oldest high-density community in Harare, established in 1907, and home to a major trading market (Mbare Musika) and bus station for intercity transport. In contrast to the other two communities, Mbare has many blocks of flats built in the 1940s as “hostels”. The capacity of those flats is greatly surpassed with occupancy rates for single rooms sometimes going up to 2-3 households The proportion of individuals reporting previous symptoms of flu-like illness was higher among those with positive SARS-CoV-2 serology compared to those with a negative serology. However, almost half of those with positive serology did not remember any symptoms in the 6 months prior to the survey. This is in line with a high proportion of asymptomatic or mildly symptomatic SARS-CoV-2 infections especially among young people ,24,25, bThose with a positive serology were more likely to have had contact with a person with confirmed SARS-CoV-2 infection and attended large events with more than 50 people – even though most of these events took place outdoors. Most households had at least intermittent access to soap and/or hand sanitisers and masks, while access to running water was available in Highfield and Mbare only.The strengths of our survey include the relatively large sample size and high household participation rates. Furthermore we used a testing methodology which has been rigorously validated on thousands of paired serum and DBS samples Our study has several limitations. While we recruited more than 95% of approached households, we only obtained DBS samples from 72·2% of potentially eligible study participants. Reasons for not being able to obtain a DBS sample was refusal by the study participant or absenteeism. Similar to other surveys, men aged 20-49 years were less likely to be included in the survey as they were away from the households for work ,21. HoweIn conclusion, we found evidence of an extremely high rate of SARS-CoV-2 infections in three high density communities in Harare following the second SARS-CoV-2 infection wave. Prevention and control measures such as contact restrictions (lockdowns and school closures), social distancing, heightened hygiene measures and improved case detection were either not sufficiently timely (i.e. too late) or effective in limiting transmission. In Mbare, the proportion of individuals with evidence of past infection was more than 60%, which is close to the community infection threshold. However, new variants and waning immunity may mean that re-infection and onward transmission of individuals previously infected is possible.Also, other more rural parts of the country are unlikely to have equally high rates of past SARS-CoV-2 infections. The third wave which seems to be disproportionally affecting the Mashonaland West province may be explained by that . OngoingGCRF, Government of Canada, Wellcome Trust, Bavarian State Ministry of Sciences, Research, and the ArtsKK, RAF, CN, HM, SR, MH conceptualised the study. AF, TB, NR, IDO collected the data in Zimbabwe. PC and KM supported field work and communication within the communities. VS lead the data analysis with input from TB and NR. AW and RRA established and validated the assay. AW oversaw laboratory testing. RRA and IP conducted the laboratory test. KK, VS, AF and RAF wrote the first draft of the manuscript. All authors provided input to the draft manuscript and read and approved the final manuscript.Individual, anonymised participant data and a data dictionary will be available through The London School of Hygiene & Tropical Medicine repository (Data Compass) 12 months after publication of results.AW declared that Roche provided reagents and machine for discounted rates for all projects involving SARS-CoV-2 serology. AW holds part of a patent application with the German Patent and Trade Mark Office relating to safe transport of DBS for analytical purpose; this application was not used in the current study. RAF received funding from the Wellcome Trust since 2017, is a member of the Zimbabwe national COVID19 Advisory Panel and a member of the CDC Africa Strategic Advisory Group of Experts for COVID19. All the other authors report no conflicts.


Bacillus cereus is widely distributed in foods, especially dairy products, and can lead to diarrhea (non-emetic B. cereus) and emesis (emetic B. cereus). Although diarrhea due to B. cereus is usually mild, emesis can lead to acute encephalopathy and even death. To develop rapid and sensitive detection methods for B. cereus in foods, specific primers targeting the gyrase B (gyrB) and cereulide synthetase (ces) genes were designed and screened using recombinase polymerase amplification (RPA). Probes and base substitutions were introduced to improve specificity and eliminate primer-dependent artifacts. The 5′ ends of the reverse primers and probes were modified with biotin and fluorescein isothiocyanate for detection of RPA products on a lateral flow strip (LFS). The developed RPA-LFS assay allows detection within 20 min at 37°C with no cross-reactivity with other foodborne pathogens. The limit of detection was 104 copies/ml and 102 CFU/ml in pure cultures and milk, respectively. Comparisons with established methods using cream obtained similar results. A specific, rapid, and sensitive RPA-LFS assay was successfully developed for on-site detection of B. cereus in dairy products to distinguish emetic from non-emetic strains. The established methods were also evaluated in cream by comparing with standard culturing method and obtained consistent results.The limit of detection was both 10-The developed RPA-LFS systems allowed detection within 20 min under 37°C with no cross-reaction with other foodborne pathogens.-Reducing Cross dimer formation and enhancing primer detection performance by introducing base mismatches.Bacillus cereus is a Gram-positive, facultatively anaerobic, rod-shaped, and endospore-forming bacterium. The B. cereus group consists of several highly homologous species, including B. cereus, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides, and Bacillus cytotoxicus , B. cereus is identified by culturing, which is both time-consuming and labor-intensive (B. cereus involves dilution of 25 g (or 25 ml) of the sample with 225 ml of diluent and homogenization, followed by serial dilution of the sample homogenate and culturing on nutrient broth plates for 24 to 48 h at 30 ± 1°C. In addition, the accuracy of the results is questionable because of the similar morphologies of bacterial colonies of different B. cereus group members  genes of emetic B. cereus by carefully designing and modifying specific primers and probes, and optimizing the reaction conditions. The detection sensitivity and specificity of the RPA-LFS assay were compared to those of standard culturing methods for the detection of emetic B. cereus in raw milk and cream. The proposed RPA-LFS assay significantly improves on-site detection of emetic and non-emetic B. cereus in dairy products.In order to meet the growing demand for fast, reliable, equipment-free technologies for on-site detection of foodborne pathogens, our group established the RPA-LFS assay for detection of the B. cereus  were used to establish the RPA-LFS assay. Ten sputum-isolated strains of non-emetic B. cereus and nine sputum-isolated strains of emetic B. cereus and other B. cereus group members, including B. thuringiensis (ATCC 10792), B. cytotoxicus (Marine Culture Collection of China [MCCC] 1A12730), B. mycoides (ATCC 6462), and B. pseudomycoides (MCCC 1A12732), were selected to evaluate the specificity of the RPA-LGFS assay. Bacillus strains  and several common foodborne pathogens  were used to assess the exclusivity of the assay.Reference strains of emetic and non-emetic B. cereus was confirmed in accordance with the National Food Safety Standard GB/T 4789.14-2014. The raw milk and cream were free of B. cereus, indicating that the detection of B. cereus was due to external addition. Spiked raw milk was used to determine the sensitivity of the RPA-LFS assay. The cream was used to detect contamination with emetic and non-emetic B. cereus bacteria and spores, and to compare the detection results of the RPA-LFS assay with those of standard culturing methods.The dairy products  used in the present study were obtained from a local market. The presence of B. cereus contamination). RPA-LFS system and qPCR were used for detection, respectively.The 50 clinical samples used in this study were all from the Lianyungang Center for Disease Control and Prevention, including commercial dairy and expired dairy  in accordance with the manufacturer’s instructions. In dairy products, gDNA extraction from vegetative bacteria included enzymatic digestion of milk proteins as described by 6 CFU/ml was used as a template in the reactions.The genomic DNA (gDNA) of emetic and non-emetic gyrB gene (GenBank accession number NC_004722.1) and ces gene (GenBank accession number DQ360825.1) were designed using Primer Premier 5.0 software . The 5′ ends of the reverse primers and probes were labeled with biotin and fluorescein isothiocyanate (FITC), respectively. The 3′ ends of the probes were blocked with a C3 spacer. The base located more than 30 bases away from the 5′ end and 15 bases away from the 3′ end was replaced with a tetrahydrofuran site. The primers and probes targeting the gyrB gene were used to specifically detect B. cereus group members among different foodborne pathogens, and the ces gene was used to identify emetic B. cereus among B. cereus group members. All primers and probes were synthesized by General Biosystems (Anhui) Co., Ltd. .Primers and probes targeting the ® Liquid DNA Amplification Kit . Each RPA reaction contained 25 μl of 2 × reaction buffer, 5 μl of 10 × Basic e-mix, 2.5 μl of 20 × core mix, 2.1 μl of each primer (10 μM), 9.8 μl of distilled water, and 1 μl of the template. To initiate the reaction, 2.5 μl of magnesium acetate (280 mM) was added to the mixture. After protein-induced DNA bending and centrifugation, the mixture was immediately incubated for 30 min at 37°C. The amplification products were purified with the MonPure™ Gel and PCR Clean Kit  and separated by electrophoresis on a 1.5% agarose gel.The RPA assay was performed in accordance with the manufacturer’s instructions of the TwistAmp® DNA Amplification nfo Kit. The RPA reaction contained 29.5 μl of rehydration buffer, 2.1 μl of forward primer (10 μM), 2.1 μl of reverse primer (10 μM), 0.6 μl of probe (10 μM), 1 μl of template, a dried enzyme pellet, and distilled water (up to 47.5 μl). To initiate the reaction, 2.5 μl of magnesium acetate (280 mM) was added to the mixture. After fully bending and brief centrifugation, the mixture was immediately incubated for 5–30 min at 30°C–42°C. Then, 5 μl of the amplification products was used for detection with an LFS . The RPA products were added to the sample pad of LFS, and the stick of the LFS was inserted into 100 μl of the sample buffer for 3 min and then visually read.The RPA assay was performed according to the manufacturer’s instructions of the TwistAmpB. cereus group members  was extracted and then tested using the optimized RPA-LFS conditions. The tested samples included ten B. cereus sputum-isolated strains, nine emetic B. cereus sputum-isolated strains, and one standard strain of each bacterium. Each experiment was performed in three triplicates.To evaluate the specificity of the RPA-LFS assay, the gDNA of B. cereus-free in accordance with National Food Safety Standard (GB/T 4789.14-2014). To assess the limit of detection (LOD) of the RPA-LFS assay for emetic and non-emetic B. cereus, gDNA extracted from emetic and non-emetic B. cereus was mixed with 108 copies/ml of L. monocytogenes gDNA and raw milk spiked with 10-fold dilutions of gDNA of emetic and non-emetic B. cereus . Each experiment was performed in triplicate.Raw milk was obtained from a local market and verified as B. cereus-free in accordance with the National Food Safety Standard GB/T 4789.14-2014 to establish artificially spiked emetic and non-emetic B. cereus models. To evaluate the lower LOD of vegetative bacteria, 25 g of cream was dissolved in 225 ml of nutrient broth, incubated with either emetic or non-emetic B. cereus to final concentrations of 100–102 CFU/ml, and then enriched at 30°C. An equal amount PBS was used as a control. Then, 1 ml of the enrichment solution was collected at 0, 2, 4, and 6 h and gDNA was extracted from bacterial cultures. The RPA-LFS assay was performed as described above. Cell counts of the contaminated food samples at each time point were determined on nutrient agar after incubation for 24 h at 30°C. For spore evaluation, 25 g of cream was dissolved in 225 ml of nutrient broth and incubated with spores of either emetic or non-emetic B. cereus to final concentrations of 103–105 CFU/ml. An equal amount PBS was used as a control. Then, 1 ml of the contaminated samples was collected and gDNA was extracted from the vegetative bacteria and spores. Cell counts of the contaminated samples were also determined. Each experiment was performed in three triplicates.Cream was purchased from a local market and verified as gyrB gene of B. cereus . Primers and probes were also designed to specifically target the pMB1 replicon rep . The results were analyzed using LightCycler® 480 Instrument Software version 1.1.1 in accordance with the Minimum Information for Publication of qPCR Experiments guidelines . A pair of specific primers targeting the conserved regions of the gyrB gene was designed, and performance was tested in the RPA reactions using the gDNA of non-emetic B. cereus. The purified RPA products were separated by electrophoresis on a 1.5% agarose gel. A 379-bp band was obtained, and no obvious primer–dimer or non-specific amplification was observed in the no template control (NTC) , other Bacillus strains , and common foodborne pathogens . The results indicated that the bands of the B. cereus group members were the expected size, thereby confirming the specificity of the gyrB primers (All strains were verified by detection of the es genes . Meanwhiol (NTC) . To scre primers  with no  primers .gyrB-P was designed based on the forward and reverse primers for gyrB. After RPA amplification with gyrB-F/R and gyrB-F/R/P, samples were added to the sample pad. The results showed that the specific primers and probes obtained bands of expected sizes with no signal for the NTC . No bands were detected for the other Bacillus strains and common foodborne pathogens  to a concentration of 108 copies/ml, and the mixed gDNA was detected with the RPA-LFS assay for emetic and non-emetic B. cereus. The results were the same as those obtained with the purified gDNA  and FITC, thereby significantly increasing the risk of false-positive results.Here, the RPA-LFS assay was developed from detection of emetic and non-emetic sitivity . The RPA. cereus . Althoug. cereus . The exa® DNA Amplification nfo Kit. The gyrB gene was used as a biomarker of B. cereus, and the ces gene was used as a biomarker of emetic B. cereus to differentiate among B. cereus group members  in pure B. cereus or with other bacterial strains or raw milk. Spiked cream samples were evaluated by comparing the detection results with standard culturing methods. The established RPA-LFS assay for emetic and non-emetic B. cereus in dairy products is rapid, simple, sensitive, and instrument-free, thereby facilitating on-site detection of emetic and non-emetic B. cereus.In this study, a novel method was established for detection of emetic and non-emetic The original contributions presented in the study are included in the article/LL, WL, and YW designed the research. LW, HuY, MZ, WZ, and KW conducted the research. HaY and YS analyzed the data. LW and JD wrote the manuscript.YW directed the project. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In recent years, the increasing incidence of pancreatic carcinoma (PC) patients has become one of the hot issues in the world. microRNAs (miRNAs) can act as oncogenes or tumor suppressor genes and have unpredictable effects on tumors, thus affecting the prognosis and survival of cancer patients. In this paper, we mainly studied the role of microRNA (miR)-409 in PC. The expression levels of miR-409 were analyzed by qRT-PCR. Kaplan-Meier curve and Cox regression were used to analyze the relationship between miR-409 and patient prognosis. The effects of miR-409 on the abilities of proliferation, migration and invasion were detected by CCK-8 and Transwell. The expression levels of miR-409 were down-regulated in PC, compared with normal controls. The prognosis of patients with low miR-409 expression is significantly poor in comparison with those with high expression. The down-regulation of miR-409 was conducive to the proliferation, migration and invasion of PC cells. miR-409 is a tumor suppressor of PC, the clinical significance of miR-409 in pancreatic cancer and related tumor cell function was clarified.  Pancreatic carcinoma (PC) is a common malignant tumor of the digestive tract ,2. Its imicroRNAs (miRNAs) usually consist of 19–25 nucleotides, with an average of 22 nucleotides ,7. ThesemiR-409 may have a crucial clinical or functional role in PC. This study aimed to investigate the prognostic value and clinical significance of miR-409 in PC using the patients’ tumor tissues. The expression levels of miR-409 in cell tissues and cell lines of PC patients were measured. Through the collected clinicopathological data, the relationship between miR-409 and the patient prognosis was also analyzed. Meanwhile, we further studied the proliferation, migration, and invasion abilities of cells, proving the important role of miR-409 in PC.A total of 143 pairs of PC tissues and adjacent normal tissues from patients were collected by surgery in the Second Affiliated Hospital of Kunming Medical University during October 2011 and October 2015. All the tissue samples collected were immediately frozen in liquid nitrogen and restored at −80°C until use. There were no patients who had received other adjuvant treatment including chemotherapy or radiotherapy before surgery and all samples were histopathologically confirmed. The clinical characteristics of PC patients were recorded in detail, including gender, age, tumor size, TNM stage, and lymph node metastasis. All patients signed the informed consent form, and the experiment was supported by the Medical Ethics Committee of The Second Affiliated Hospital of Kunming Medical University. The time of the initial operation was recorded and the patients were followed up regularly for 5 years.2.The human PC cell lines, PANC-1 and SW1990, BxPC3 and AsPC-1 were purchased from the American type culture collection, the human pancreatic ductal epithelial cell line (HPDE) was purchased from the Cell Repository, Chinese Academy of Sciences . All cell lines were routinely cultured in RPMI 1640 medium  and 10% fetal bovine serum  were supplemented into the mixture. All cells were cultured at 37°C with 5% COSix-well plates were used for cell transfection. Based on the mature sequences of miR-409, miR-409 mimics (5ʹ- GAAUGUUGCUCGGUGAACCCCU-3ʹ), miR-409 inhibitors (5ʹ-AGGGGUUCACCGAGCAACAUUC-3ʹ), and corresponding negative controls  were purchased from RiboBio, Co., Ltd.  and transfected into the cells according to the manufacturer’s using Lipofectamine 2000 .−ΔΔCt method by normalized to U6 snRNA. In this experiments, Reaction conditions were 95°C for 4 min, followed by 35 cycles of 94°C for 30 sec, 58°C for 20 sec, and 72°C for 20 sec. Primer sequences were miR-409 forward: 5ʹ-GCCGAGAGGGGTTCACCGAG-3ʹ and reverse: 5ʹ-GCAGGGTCCGAGGTATTC-3ʹ; and U6 forward: 5′-CTCGCTTCGGCAGCACA-3′; and reverse: 5′-AACGCTTCACGAATTTGCGT-3′.Total RNA was extracted from PC tissue using TRIzol reagent . The concentration and purity of RNA were measured by NanoDrop 2000 . Then, miRNA was reverse transcribed to cDNA using the TaqMan miRNA reverse transcription kit . The following Real-time quantitative PCR (qRT-PCR) were carried out in triplicate by biosystems 7500 apparatus and SYBR-Green I Master mix kit  was used to evaluate the amount of PCR products. miR-409 levels were calculated by the 23 cells/well) were seeded into 96-well plates and cultured overnight. Cells were treated with CCK-8 reagent at 0, 24, 48, and 72 hours. The absorbance was measured at 450 nm, and the value represented the proliferation capacity of CCK-8. All experiments were repeated three times.The Cell Counting Kit-8   was used5 cells in 200 μL of serum-free medium were placed in the upper chamber and the upper chamber was not coated with Matrigel . The lower chamber was filled with 500 μl medium containing 10% FBS. Then, 4% paraformaldehyde solution was used to fix the cells, 0.1% crystal violet was used to stain the cells.The Transwell chambers used in the cell migration and invasion experiments  were 8-μThe difference between the cell invasion experiment and the migration experiment was that Matrigel was precoated on the bottom of the upper chamber before the cell invasion experiment. The other steps were the same.www.targetscan.org), miRDB (mirdb.org), and CancerMIRNome  were used to explore the potential target genes and the binding sites of miR-409. The potential target genes were filtered from the three databases using the Venny tool (Venny 2.1.0) and listed in Supplementary Three online bioinformatics databases, including TargetScan  to construct the GAB1 3ʹ-UTR WT or GAB1 3ʹ-UTR MUT vectors. SW1990 and BxPC3 cells were seeded in 96-well plates and cultured for 24 h. Then cells were transfected with GAB1 3ʹ-UTR WT or GAB1 3ʹ-UTR MUT and miR-409 mimics, inhibitors, or NCs using Lipofectamine 2000  for 24 h. Then, a dual-luciferase reporter assay system  was used to measure the fire luciferase activities that were normalized to Statistical analyses were performed with SPSS version 22.0 software  and GraphPad Prism 5.0 . Student’s t-test and one-way analysis of variance (ANOVA) test were used to test statistical significance. All results were expressed as the mean ± standard deviation (SD). The patient prognostic value was estimated by the Kaplan-Meier curve. Factors related to the overall survival were assessed using Cox regression. It was considered statistically significant when the value of P was less than 0.05.The study was carried out to explore the clinical significance and functional role of miR-409 in PC. RT-PCR was used to detect the expression levels of miR-409 in PC tissues and cells. Chi-square test was used to analyze the association between miR-409 expression and clinical parameters of patients, Kaplan-Meier method and Cox regression analysis were performed to explore the clinical role of miR-409 in PC. CCK-8 assay, Transwell assay, and Dual-luciferase reporter assay were carried out to assess the functional role of miR-409 in PC cells.P < 0.001). miR-409 expression in PC cell lines  was also significantly lower than that in normal cell lines (HPDE)  and TNM staging (P = 0.004). But, the expression of miR-409 was not significantly correlated with age, gender, location, carcinoma embryonic antigen (CEA), CA199, tumor size and differentiation (P > 0.05).To facilitate follow-up studies, 143 PC Patients were divided into two groups by the median miR-409 expression, including 71 cases in the high expression group and 72 cases in the low expression group. P = 0.032). To obtain the independent factors affecting the patients’ prognosis, the multivariate Cox regression analysis on the 5-year survival was performed. The results confirmed that miR-409 expression , lymph node metastasis  and TNM stage  were the independent prognostic indicators of PC patients .Cell proliferation, migration, and invasion are crucial to tumor progression, so it is necessary to investigate the effect of miR-409 expression level on tumor cellular functions. The expression levels of miR-409 in SW1990 and BxPC-3 cell lines were lower than those of the other two PC cell lines. Therefore, SW1990 and BxPC-3 cell lines were selected as the research objects for tumor cellular functions experiments. qRT-PCR was used to detect the expression levels of miR-409 after cell transfection. The results indicated that miR-409 mimics could increase the expression levels of miR-409, while miR-409 inhibitors could decrease its expression levels, compared with mimic/inhibitor NC .To explore the molecular mechanisms underlying the function of miR-409 in PC, bioinformatics analysis and dual-luciferase reporter assay were used to predict the putative targets of miR-409. Among these targets that were predicted using bioinformatic analysis , GAB1 waStudies have shown that miRNAs are inextricably associated with tumors. Different types of cancer have different miRNA expression profiles, which can distinguish adjacent normal tissues . The dismiR-409, located on the chromosome region 14q32, has been found to play a tumor suppressor role in a variety of tumors ,26. In tAnalyzing the clinical characteristics of PC patients, low miR-409 expression is closely related to the high TNM stage and positive lymph node metastasis. The lymph node metastasis and TNM stage are important prognostic indicators of tumor patients. Besides, patients with low miR-409 expression have also been shown to have a poor prognosis. Based on the above studies, miR-409 may have a prognosis predictor value of PC patients. Similar results have been reported for other cancers. For example, in breast cancer, the decreased level of miR-409 expression is related to the prognosis of patients, which may be a promising predictor of breast cancer recurrence .Proliferation, migration and invasion of tumor cells are important processes in the development of cancer ,29. For In conclusion, the expression of miR-409 was down-regulated in PC. PC patients with low miR-409 expression usually have a poor prognosis. The low miR-409 expression may accelerate the tumor cell progression of PC by targeting GAB1. miR-409 may have important clinical significance for the prognosis of patients with PC, providing new ideas and bringing new hope for the treatment of PC.Click here for additional data file.


Background: Previous studies have demonstrated that serum N-terminal proB-type natriuretic peptide (NT-proBNP) was a predictor of adverse cardiovascular outcomes after surgery. We performed a prospective study to evaluate if NT-proBNP could be a sensitive marker of overall postoperative outcomes in older patients undergoing major elective non-cardiac surgery when combined with myoglobin (MYO).Methods: Two hundred and three adults aged ≥65 years were enrolled in the study. The American Society of Anesthesiologists (ASA) physical status of patients were I to IV. Blood samples would be taken before and 2 h after the surgery for each patients and NT-proBNP and MYO concentrations (NT-proBNP baseline/ 2 h and MYO baseline/ 2 h) of these samples would be measured immediately. The primary outcome was moderate to severe complications, which were based on the Clavien–Dindo Classification (CDC) scheme (≥CDC grade 3), and the secondary outcomes were major complications within 30 days after surgery. This study was registered at China Clinical Trial Registry .Results: Overall, moderate to severe complications occurred in 15 patients (7.4%) and major complications occurred in 18 patients (8.9%). Both preoperative and postoperative NT-proBNP values were independent predictors of moderate to severe complications (area under the curve (AUC), 0.820; 95% CI: 0.728, 0.912, P < 0.001; AUC, 0.785; 95% CI: 0.685, 0.885, P < 0.001). When NT-proBNP baseline and MYO-2 h were combined (NT-proBNP baseline × MYO-2 h), the predictive power was improved .Conclusions: A combination of perioperative NT-proBNP and postoperative MYO concentrations was a good predictor of postoperative complications in elderly patients who underwent major non-cardiac surgery. Using fast and dynamic tests provided by point-to-care-testing, NT-proBNP and MYO concentration measurements provided useful guidance for therapy before or soon after surgery, thus helping to reduce postoperative complications in elderly patients. Postoperative death has been ranked as the third main cause of death globally . AlthougMyoglobin (MYO) is a structural protein in the cytoplasm of myocardial and skeletal muscle cells that can be rapidly released into the blood once damage occurs , 16. MYOTherefore, the present study investigated whether serum NT-proBNP and MYO concentrations could predict mortality and morbidity after major elective non-cardiac surgery in elderly patients. We also determined the clinical prognostic value when these two markers were combined. Considering the pathophysiological changes that occur during surgery, which is a composition of surgical trauma and preoperative functional response, we assumed that the combination of these two biomarkers might provide a better predictive value because NT-proBNP and MYO concentrations are associated with functional challenges and tissue damage, respectively.https://www.chictr.org.cn). This single-center, prospective observational cohort study was performed from June 2020 to November 2020. Patients eligible for inclusion were scheduled for major non-cardiac surgery (>3 h) under general anesthesia, aged ≥65 years, and had an ASA of I–IV. There was no limitation regarding gender. Patient exclusion criteria included known hematological diseases, long-term use of sedative or psychotropic drugs, an unstable mental state, and a history of allergy to anesthetics. Written informed consent was obtained from all patients or a close relative.Approval for this study  was provided by the Ethics Committee on Biomedical Research at West China Hospital of Sichuan University, Sichuan, China (Chairperson Prof Shaolin Deng) on May 25, 2020 and registered at the China Clinical Trial Registry  in the operating room. General anesthesia was induced by intravenous propofol 1.5–2.5 mg/kg, and sufentanil 3 μg/kg, cisatracurium 0.2 mg/kg or vecuronium bromide 0.1 mg/kg was subsequently administered. After tracheal intubation, the patients were ventilated to normocapnia using an inspired oxygen fraction of 0.5 with a fresh gas flow of 2 L/min of oxygen and air. Anesthesia was maintained with an intravenous remifentanil infusion and a sevoflurane or desflurane inhalation or propofol TCI. A target BIS value of 40–60 was maintained by adjusting the dosage of the general anesthetics. At skin closure after surgery, desflurane and remifentanil were terminated. Neostigmine 20 μg/kg with atropine 10 μg/kg was administered to antagonize the residual neuromuscular block unless contraindicated. Patients were transferred to the postanesthesia care unit (PACU) or ward after tracheal extubation, which was performed after patients were confirmed to be responsive to verbal commands and to have adequate spontaneous respiratory responses. Some patients were transferred into the intensive care unit (ICU) directly. All the anesthetic or surgical procedures were administered at the discretion of the attending anesthetist or surgeon, respectively.All patients underwent venous blood sampling two times to measure NT-proBNP and MYO concentrations, just before surgery in the operating room (NT-proBNP baseline/MYO baseline) and 2 h after surgery (NT-proBNP−2 h/MYO−2 h). Blood samples were collected in EDTA evacuated tubes, immediately centrifuged, and then analyzed using a Finecare 3 plus FS-205 immunofluorescence automatic analyzer  in the operating room. The normal reference ranges were 0–300 pg/ml and 0–58 ng/ml for NT-proBNP and MYO, respectively.www.assessurgery.com) to calculate the comprehensive complication index (CCI) for each patient.Research personnel followed patients throughout their hospital stay once a day to ascertain the presence of postoperative complications. The severity of complications was categorized using a modified CDC scheme . Based oAfter hospital discharge, patients were contacted by telephone 30 days after surgery to confirm the incidence of post-discharge complications. Postoperative major complications were based on a visit during their hospital stay and telephone follow-up after discharge. Mortality was defined as death within 30 days after surgery. A stay in the ICU for > 24 h was also considered to be a major complication. Acute kidney injury (AKI) was defined according to kidney disease improving global outcomes (KDIGO) criteria (2012) . CardiovThe sample size calculation was based on comparing the area under the curve (AUC) of the receiver-operating characteristic (ROC) curves using MedCalc (Version 19). Assuming an outcome event rate of 8% (based on our previous study), a moderately good AUC of 0.75 for blood NT-proBNP or MYO concentrations, a sample size of 188 patients had 90% power to detect a clinically relevant difference in AUC values . To account for 10% of patients who may have been lost to follow-up, we aimed to recruit a total of 206 patients to the study. The missing data were excluded.P < 0.05. Comparison of interval data was performed using the independent sample t-tests, the Mann–Whitney, or the Kruskal–Wallis tests when appropriate. NT-proBNP/MYO concentrations and NT-proBNP × MYO concentrations were entered as both continuous and categorical variables at different levels for analyses. The ROC curve analysis was performed to identify the predictive values of NT-proBNP and MYO concentrations for postoperative complications. The performance of continuous variables was analyzed with ROC curves, with the AUC being calculated. Cutoff values were evaluated based on the Youden index. Relative risk (RR) and 95% confidence intervals (CIs) were determined using a chi-squared test.Statistical analysis was performed using SPSS Version 15 software . All statistical tests were two-sided, and significance was assumed at a value of Between June 2020 and November 2020, 603 patients met the study inclusion criteria. Only 224 patients provided consent to participate in the study, and 12 patients had their surgery unexpectedly canceled. Finally, 212 patients in our hospital were enrolled in the study and 203 (96.5%) completed their 30-day follow-ups.According to our protocol, blood samples were taken both in the operating room before surgery and in the PACU 2 h after surgery for each patient. For those patients transferred directly into the ICU or wards, without entering PACU, blood samples were taken there.The median age of included patients was 72 years  of which 26.6% were female. The demographic characteristics and preoperative comorbidities of the patients are shown in P < 0.01). No difference was found in the perioperative NT-proBNP mean concentration among complication-positive or negative patients (P > 0.05). The levels of MYO at 2 h in patients who developed ≥CDC grade 3 were higher than in patients without corresponding complications (P < 0.01). But for patients who developed major complications, there was no difference in the MYO baseline, MYO at 2 h, or changes in the MYO concentration. The CCI score was lower in patients with reference concentrations of NT-proBNP or MYO than in those with abnormal concentrations (P < 0.005).As shown in P ≤ 0.001). The cutoff concentration of the NT-proBNP baseline was 312 pg/ml and it exhibited the best combined sensitivity (0.733) and specificity (0.818). The cutoff concentration of NT-proBNP at 2 h was 340 pg/ml, the sensitivity and specificity of which were 0.733 and 0.802, respectively. The AUC of combination NT-proBNP and MYO using NT-proBNP baseline × MYO at 2 h was 0.841 . The cutoff of NT-proBNP baseline × MYO at 2 h was 32,828 and had the best combined sensitivity (0.800) and specificity (0.797). For major complications, the AUC of the BNP baseline and the NT-proBNP concentration at 2 h were 0.794 (P ≤ 0.001) and 0.745 (P = 0.001), respectively. The results of ROC analysis are shown in The ROC analysis showed the predictive value of NT-proBNP baseline concentrations, NT-proBNP and MYO at 2 h for complications ≥grade 3, or major complications ; Table 3P < 0.001) and the RR of the NT-proBNP baseline ≥600 pg/ml was 1.379 ; the RR of the NT-proBNP baseline × MYO-2 h between 30,000 and 60,000 was 2.488  and the RR of the NT-proBNP baseline × MYO-2 h ≥60,000 was 7.092 . For major complications, the RR of the NT-proBNP baseline between 300 and 600 pg/ml was 3.257  and the RR of the NT-proBNP baseline ≥600 pg/ml was 3.861 ; the RR of the NT-proBNP baseline × MYO−2 h ≥60,000 was 6.230 .Relative risk values were also calculated to identify the risk of different concentrations of the NT-proBNP baseline and the NT-proBNP baseline × MYO-2 h . For comThe present study demonstrated that preoperative the NT-proBNP concentration was a valuable predictor of postoperative moderate-to-severe complications and also major complications. When combined with postoperative MYO concentration measurements, the predictability was further improved. Our study extends the findings of other cohort studies in this research area, most of which focused on the relationship between preoperative NT-proBNP concentrations with associated postoperative MACE or mortality , 9, 24. In the present study, the AUC of the NT-proBNP baseline and NT-proBNP baseline × MYO-2 h were 0.820 and 0.841, respectively, in predicting moderate to severe complications. The previous study of Payne et al. found the AUC was 0.792 in predicting all-cause mortality 1 year after major non-cardiac surgery in 345 patients . In anotAccording to a previous study, an elevated concentration of plasma NT-proBNP before or soon after thoracic surgery (1 h) for lung cancer was a strong independent predictor for the occurrence of atrial fibrillation . HoweverAnother interesting finding in our study was that the AUCs of the MYO baseline and MYO-2 h were 0.505 and 0.690, respectively for complications ≥CDC grade 3. Contrary to our expectations, MYO alone had no value in predicting postoperative complications. In fact, previous studies have proven that MYO level was a potential biomarker for evaluating sepsis severity and poor outcomes, and the area under the ROC curve was 0.824 . Also, tP = 0.746), which suggested that patients with NT-proBNP baselines ≥600 had a lower risk than other patients. But we thought the predictive power was underestimated because the sample size was relatively small. Also, the joint predictive power of the NT-proBNP baseline and MYO-2 h may have been underestimated. In addition, as the sample size was relatively small, the number of patients with events (moderate to severe complications or major complications) was also small. Large sample studies are needed to confirm the outcomes of the present study.One limitation of the present study was the RR value of NT-proBNP baseline ≥600 for CDC ≥ 3 was 1.379  was provided by the Ethics Committee on Biomedical Research, West China Hospital of Sichuan University, Sichuan, China (Chairperson Prof Shaolin Deng) on 25 May 2020 and registered at China Clinical Trial Registry. The patients/participants provided their written informed consent to participate in this study.YZ: conceptualization, methodology, and writing the original draft. XH: conceptualization, methodology, and software. YZ and MC: visualization and investigation. MO: data curation. TZ: supervision. All authors contributed to the refinement of the study protocol and approved the final manuscript.This study was supported by the National Key R&D Program of China  (Grant No. 2018YFC2001800); the CAMS Innovation Fund for Medical Sciences (grant number 2019-I2M-5-011); the Science and Technology Department of Sichuan Province, China (Grant No. 2020YJ0052); the China Postdoctoral Science Foundation (grant number 2019M663507); the Post-Doctor Research Project, West China Hospital, Sichuan University (project number 19HXBH064); the National Clinical Research Center for Geriatrics, West China Hospital of Sichuan University (Grant No. Z2018A02); and the 135 Project for Disciplines of Excellence, West China Hospital, Sichuan University (Grant No. ZYJC18010).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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The temperature dependence of the excitonic photoluminescence (PL) and nonlinear absorption characteristics of CdTe nanocrystals (NCs)/polyvinyl alcohol (PVA) film are investigated using steady-state/time-resolved PL spectroscopy and Z-scan methods. The excitonic PL peaks of CdTe NCs can be observed at the wavelengths from 560 to 670 nm, with size changes from 2.1 to 3.9 nm. From the temperature-dependent PL spectra, the smaller photon energy of the PL emission peak, the rapidly decreasing PL intensity, and the wider linewidth are observed with increasing temperature from 80 to 300 K. It is revealed that the excitonic PL is composed of both trapped state and band-edge excitonic state, which presents biexponential fitting dynamics. The short-lived species is due to the surface-trapped state recombination in NCs, which has a photogenerated trapped channel and a time-resolved peak shift. The species with a long-lived lifetime is ascribed to the intrinsic excitonic recombination. Through the femtosecond Z-scan method, the nonlinear absorption coefficient becomes smaller with the increase in the size of the NCs. The optical properties of the CdTe NC/PVA film show the potential of II-VI traditional NCs as display and luminescent materials that can utilize the combination of excitonic PL and nonlinear absorption for expanded functionality. Polymer nanofilms are versatile, functional materials in which nanoscale 1–100 nm) inorganic nanoparticles are dispersed in an organic polymer matrix to display enhanced optical, magnetic, and mechanical characteristics –100 nm i. The incIn our investigation, we discuss the temperature-dependent, steady-state, and time-resolved PL properties of CdTe NC/PVA films compared with pure CdTe NCs. The temperature dependence on steady-state PL peak energy, intensity, and linewidth of the NCs are investigated. The exciton binding energy, optical phonon-exciton coefficient, and longitudinal optical-phonon energy are calculated by fitting to the temperature-dependent excitonic PL spectra for CdTe NC/PVA films. As size decreases, the nonlinear absorption becomes bigger, which is ascribed to the enhancement of the quantum confinement effect.2·2.5H2O was added in 500 mL water, and appropriate thioglycollic acid was dissolved under stirring, followed by changing the pH via the dropwise addition of NaOH solution with 1 mol/L concentration. The obtained solution was transparent and placed in a three-necked flask fixed with a division plate and valves, then deaerated with nitrogen bubbling for 30 min. The prepared 0.612 mL NaHTe was added to the solution together with a slow N2 gas flow under stirring. The formation and growth of the NCs progressed upon refluxing at 100 °C with a condenser attached under open conditions [Luminescent colloidal CdTe NCs are usually synthesized by reproducible one-pot ,18, precnditions . Tuning The UV–visible absorption and luminescence spectra of the synthesized CdTe NCs were analyzed on a UV–Vis spectrometer  and spectrofluorometer . A series of CdTe NC/PVA films on BK7 glass substrate were fabricated for the temperature-dependent PL measurement. A continuous-wave (CW) laser  was used as an excitation light source at 405 nm for steady-state PL measurement. The steady-state PL spectrum was collected by a charge coupled device (CCD)  with a spectrometer . The temperature-dependent PL measurement was performed using a vacuum cryostat  with the ability to realize temperature variation from 78 to 300 K. Time-resolved excitonic PL spectra were measured with an intensified CCD  with a spectrometer , of which time resolution is ~2 ns. Through second-harmonic generation, a femtosecond regenerative amplifier  produces 800 nm laser pulses with 1 kHz repetitive frequency, which are used to change the pulse wavelength at 400 nm as excited pulses.0z was about 1.6 mm, which satisfies the thin-medium condition [Z-scan setup under femtosecond laser excitation has been described previously . The opeondition . The inc2 mW/cm2; the intensity decreased to 92% after 6 h laser radiation. However, PL intensity of CdTe NC/PVA film dramatically reduced for the beginning 1 h and then reduced slowly under the same experimental conditions. The PL peak position had no shift, which suggests the thermal accumulation can be ignored below the laser fluence of 2.5 × 102 mW/cm2. With the laser fluence increasing, the thermal effect can lead to the increase in temperature in the composite; thus, the redshift of the emission peak happened at higher temperature [The PL intensity of CdTe NC/PVA 2# film under CW laser excitation, as a function of radiation time, is shown in perature .To further study the excitonic recombination of a series of CdTe NC/PVA films, the measurement of steady-state PL was performed. The temperature-dependent steady-state PL spectra of CdTe/PVA 3# are measured and shown in gE(0) is the bandgap at 0 K, α is the temperature-dependent coefficient, and β is a fitting parameter that is related to the Debye temperature. −4 eV/K, which is very close to the value of bulk CdTe (0.3 × 10−4 eV/K) [I0 is the initial luminescent intensity, and exciton binding energy (BE) corresponds to the minimal energy necessary to promote the carrier to the unbound energy level. The term BTk represents the thermal perturbation, which has a value of 26.0 meV at room temperature. BE is calculated from Equation (2) by fitting the experiment data 62.7, 54.4, 70.2, and 62.3 meV for a series of CdTe NC/PVA films, respectively, as shown in Γ0 is the inhomogeneous band broadening, and σT relates to the homogeneous broadening contribution, which arises from acoustic phonon scattering through the deformation potential with the carrier-acoustic phonon coupling intensity of σ. opΓ is connected with the optical phonon–exciton contribution to the band linewidth broadening, while opћω is longitudinal optical–phonon energy, in which ћ is the reduced Planck constant and opω is the angular frequency of optical phonon oscillation. When the temperature increases, the optical phonon contribution dominates and the band linewidth increases almost linearly with temperature change [For traditional II-VI group semiconductor NCs, with increasing temperature, the PL peak position shifted to a larger photon energy band . The tem−4 eV/K) . The β v−4 eV/K) , due to −4 eV/K) . The fit−4 eV/K) . The teme change . The firτ1 and τ2 are lifetimes of the long- and short-lived excitonic emission species, respectively. A expresses the background of the signal. B and C are the relative intensities of the two species, respectively. The width of the instrumental response function (IRF) τFWHM is recorded at the emission wavelength of the excitation laser pulse. IRF is considered with Gaussian function to convolute with the PL dynamics. The average PL lifetime of the NCs can be calculated asTo further reveal the mechanism of excitonic recombination, we carried out time-resolved PL measurement, and some results are shown in τ1) unchanged with the increasing temperature, while the lifetime of excitonic state (τ2) became longer, from 0.5 ns to 2.3 ns, when the temperature increased from 80 to 320 K. With increasing temperatures, thermal motion becomes more active and thus prevents the recombination of excitons, resulting in an increased PL lifetime [With increasing temperature, the lifetimes of the long- and short-lived species for CdTe NC/PVA films are calculated by double-exponential fitting, as shown in lifetime . Here, tlifetime . We adop−3 cm/GW) [−2 cm/GW) [The nonlinear absorption of NCs in the off-resonant spectral region is important for the potential application in optoelectronic devices . In orde3 cm/GW)  and CdSe2 cm/GW) . No signIn conclusion, the temperature-dependent spectral properties of CdTe NC/PVA films are studied via PL spectroscopy and Z-scan techniques. From the temperature-dependent PL spectra, a smaller photon energy of the PL emission peak, a rapidly decreasing PL intensity, and a wider linewidth are observed with temperature increasing from 80 to 300 K. It is revealed that the excitonic PL consists of band-edge state and trapped state emissions and, therefore, presents double exponential dynamics. The short-lived emission species is attributed to surface-trapped state recombination in NCs that has a photogenerated trapped channel and a time-resolved peak shift. The species with a long-lived lifetime is ascribed to the excitonic band-edge recombination. Through the femtosecond Z-scan method, the calculated nonlinear two-photon absorption coefficient becomes smaller as the size of the NCs increases. The temperature dependence of the PL for CdTe NC/PVA films reveals the luminescent and thermal behavior, which help us to understand CdTe NC/PVA films, for potential use in display and LED applications.


Escherichia coli cells of the proteins specified by the following type II TA modules: mazEF, chpBIK, mqsRA, and rnlAB. We visualized the localization of these proteins by fusing them with the fluorescent protein mCherry using recombinant DNA technology. We used fluorescence microscopy and image analysis software to obtain and quantify protein distribution data. With the exception of the chpBIK TA module, we found that the localization of each toxin-antitoxin complex was different from the localization of the toxin itself. Our results demonstrate clearly that the presence of the antitoxin shifts the localization of its respective toxin toward the middle of the cell, which could contribute to the reduction of cellular toxicity.Bacterial toxin-antitoxin (TA) systems encode a toxin and an antitoxin that counteracts the toxin. Such TA systems are found abundantly on bacterial chromosomes and on extrachromosomal genetic elements. The toxin is always a protein. Based on the nature of the antitoxin (protein or RNA) and on their mode of regulation, they are classified into six groups (I to VI). In the group II TA systems, both the toxin and the antitoxin are proteins, and the gene specifying the antitoxin precedes the gene specifying for the toxin. Here, we studied the intracellular localization in  Bacterial toxin-antitoxin (TA) systems are unique genetic elements that consist of pairs of genes, one encoding a toxin and one encoding an antitoxin. These systems are found abundantly, either on extrachromosomal elements, such as plasmids and bacterial phages, or on the bacterial chromosome itself. To date, toxin-antitoxin systems are divided into six types . The mosEscherichia colimazEF module (mazEF module encodes the antitoxin MazE and the toxin MazF (mazEF system is triggered by various stressful conditions (relA-encoded protein (RelA) (2O2)  ; (ii) inn (RelA) ; (iii) Dn (RelA) , as welln (RelA) ; (iv) oxA) (2O2) ; and (v)A) (2O2) . All of xin MazF . It has xin MazF . MazF haxin MazF .mazEF is the E. coli chromosomal system chpBIK. This system consists of a pair of genes, chpBI and chpBIK, which encode the antitoxin ChpBI and the toxin ChpBK, respectively . This cleavage of mRNAs in the E. coli cells leads to a reduction of protein synthesis . This was done in E. coli MC4100 +relA cells with the mcherry gene, located on plasmid pBAD18 and induced by arabinose. As can be seen in E. coli MC4100 +relA cells.Initially, we studied the intracellular localization of the protein products of the 10.1128/mBio.01417-21.2FIG S1Escherichia coli MC4100 +relA strain. mCherry is dispersed homogeneously in the cells. Download FIG S1, TIF file, 2.9 MB.Intracellular localization of mCherry in the Copyright © 2021 Mager et al.2021Mager et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the mazEF toxin-antitoxin module in E. coli cells, we fused each of these products  to the fluorescent reporter mCherry. For this purpose, primers were generated to produce mazF, mazE, and mazEF genes lacking stop codons and fused to a linker of three alanine residues, as described in Materials and Methods. The fused genes thus generated were first amplified by PCR and then cloned into the pBAD18-mcherry plasmid. The primers used for cloning are listed in the supplemental material. The cloning protocols are described in detail in Materials and Methods. The constructed fusions are illustrated schematically in To determine the intracellular localization of the protein products of the 10.1128/mBio.01417-21.3FIG S2mazE-mcherry, mazF-mcherry, chpB-mcherry, chpBK-mcherry, mqsR-mcherry, mqsA-mcherry, rnlA-mcherry, and rnlB-mcherry fusions, the gene of interest lacking a stop codon is fused to mcherry through a 3-alanine linker. In the cases of mazEF-mcherry and chpBIK-mcherry fusions, the gene sequences of mazF and chpBK, lacking their respective stop codons, are fused with mcherry through a linker of 3 alanines. Download FIG S2, TIF file, 1.2 MB.A schematic representation of the fusions constructed in this work. In the cases of Copyright © 2021 Mager et al.2021Mager et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the E. coli MC4100 +relA cells carrying the pBAD18 plasmids with the constructed fusions were grown as described in Materials and Methods. After induction with 10 mM arabinose, cell viability was determined. mazEF TA module on bacterial viability. Overproduction of the MazF-mCherry fusion caused loss of cell viability . In fact, in 20 of 37 cells of this representative image (which shows one out of 15 similar experiments), MazF-mCherry was localized at the pole of the E. coli cells  in the pole , lane 1.In contrast to the calculated position of the toxin MazF by itself , a complmazEF proteins with 10 mM arabinose. Similar results were obtained by the use of 5 mM arabinose .To support our discovery that the interaction of MazE with MazF causes a shift in the localization of MazF from the poles toward the center of the mazEF TA system protein products are unique, or whether they are similar to those of its homologue, the chpBIK TA system. As we had done for the mazEF system, to examine the intracellular localization of the protein products of the chpBIK TA system, we fused the fluorescent reporter mCherry to the toxin ChpBK, to the antitoxin ChpBI, and to the combined ChpBIK. Constructed fusions are illustrated schematically in We asked if the localization patterns of the mazEF, the TA module chpBIK is not involved in cell death, but rather inhibits E. coli cell growth . In another subpopulation of the cells, the toxin MqsR was localized between the pole and the middle of the cell (indicated by white arrows).mazEF and chpBIK TA modules, in which the gene for the antitoxin precedes the gene for the toxin, in the mqsRA TA module, the mqsR toxin gene precedes the mqsA antitoxin gene. To recreate the conditions under which the toxin is fused with mCherry and is expressed in the presence of the overproduced antitoxin, we needed to use a different approach than the one described above for the mazEF and chpBIK modules. We cloned the mqsA antitoxin gene to pBAD33 and transformed this newly constructed plasmid into E. coli MC4100 +relA cells harboring the pBAD-18-mqsR-mcherry plasmids. The cloning protocol is described in details in Materials and Methods. E. coli MC4100 +relA cells. As shown, there are still two main subpopulations of the cells. In the first, the toxin MqsR is located at the middle of the cell (indicated by yellow arrows). In the second, the toxin MqsR forms foci between the pole and the middle of the cell (indicated by white arrows). Determining the relative distribution of the foci allowed us to clarify the difference between the localization of the toxin MqsR by itself or in the presence of the overproduced antitoxin MqsA . Similarly, the antitoxin RnlB fused with mCherry also formed foci at the poles in most of the cells  would have the same pattern of the intracellular localization. We were surprised to discover that each of the TA modules studied here had its own pattern of localization for the toxin, the antitoxin, and the toxin-antitoxin complex. However, with the exception of the mazEF proteins, they may interact with mazEF-specific components, such as the peptide EDF(NNWNN) (Future experiments should provide explanation of why the toxins tend to migrate toward the poles and the reason for the shift toward the center of the cell by the antitoxin. Does this have anything with the endoribonuclease activity? Interaction with actin-like cytoskeleton proteins? Interaction with ClpXP? In the case of the F(NNWNN)  and compF(NNWNN)  . (A) MazF (in red) generates leaderless mRNAs (in green) by cleaving at ACA sites at or closely upstream pf the AUG start codons of some specific mRNAs. (B) MazF also removes the 3′-terminal 43 nucleotides of the 16S rRNA cleaving at an ACA site, thereby removing the anti-Shine-Dalgarno sequence. (C) As a result, an alternative translation machinery is generated, which is responsible for the selective synthesis of specific proteins. Download Copyright © 2021 Mager et al.2021Mager et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Primers for cloning were obtained from Integrated DNA Technologies . Other materials and suppliers were as follows: PrimeSTAR HS DNA polymerase from TaKaRa, Japan; T4 ligase, Arctic phosphatase, and restriction enzymes XbaI, SacI, EcorI, and HindIII from Thermo Scientific ; plasmid miniprep kit from Qiagen ; Wizard SV gel and PCR clean-up system from Promega; and FM 1-43FX membrane stain from Thermo Scientific .E. coli MC4100 +relA strain  pBAD18-mCherry cloning. The genes encoding the proteins MazE, MazF ChpBI, ChpBK, MqsR, MqsA, RnlA, and RnlB (lacking stop codons) were amplified by PCR from E. coli MC4100 relA+ genomic DNA. For this purpose, we used the primers that introduced EcoRI (forward primer) and SacI (reverse primer) restriction sites and a gene sequence encoding a three-alanine linker. We used primers that introduced EcoRI (forward primer) and SacI (reverse primer) restriction sites and a gene sequence encoding a linker of three alanine residues. The resulting amplicons were digested with EcoRI and SacI and ligated into pBAD18-mcherry plasmids. The following DNA fusions were constructed: mazE-mcherry, mazF-mcherry chpBI-mcherry, chpBK-mcherry, mqsR-mcherry, mqSA-mcherry, rnlA-mcherry, and rnlB-mcherry. In the protein products of these DNA fusions, mCherry was fused to the C terminus of the protein of interest through a three-alanine linker. In the case of the combined antitoxin and toxin MazEF-mCherry fusion, the gene sequence of the mazEF operon (lacking the stop codon of mazF) was amplified by PCR and cloned into pBAD18-mcherry plasmid as described above. The following protein products were generated: MazE (by itself without mCherry) and MazF-mCherry, where mCherry was fused to the C terminus of the toxin MazF through a three-alanine linker. In the case of the combined antitoxin and toxin ChpBIK-mCherry fusion, the gene sequence of the chpBIK operon (lacking the stop codon of chpBK) was amplified by PCR and cloned into the pBAB18-mcherry plasmid as described above. The following protein products were generated: ChpBI and ChpBK-mCherry. mCherry was fused to the C terminus of the toxin ChpBK through a three-alanine linker. The primers for cloning and constructed plasmids are listed in the supplemental material.(ii) pBAD33 cloning. In addition to the constructions using pBAD18, gene sequences of mazE, mqsA, and rnlB (lacking stop codons) were amplified by PCR from E. coli MC4100 genomic DNA using the primers that introduced SacI (forward) and XbaI (reverse) sites and a three-alanine linker. The resulting amplicon was cloned into the pBAD33 plasmid. The primers for cloning and constructed plasmids are listed in the supplemental material.E. coli MC4100 relA+ strain using electroporation. Single colonies were grown overnight in 10 ml of LB medium with 100 μg/ml ampicillin at 37°C and 220 rpm. A 200-μl aliquot of overnight culture was subcultured (1:20 dilution) in 10 ml of LB in the presence of 100 μg/ml ampicillin. The cells were grown at 37°C and 220 rpm to an optical density at 600 nm (OD600) of 0.4. To express protein-mCherry fusions, 10 mM arabinose was added to the cell culture, and the cells were incubated at 37°C for 4 h.Cloned plasmids were transformed into the Cells were harvested by centrifugation at 14,000 rpm for 2 min and washed twice with phosphate-buffered saline (PBS). For labeling, cell membrane cells were resuspended with 20 μl of FM-143FX dye. Then, for fixation, 100 μl of 4% formalin was applied for 15 min, and the cells were washed twice with PBS (pH 7.2) and resuspended in 10 μl of PBS and 10 μl of polyvinyl alcohol (PVA). Aliquots (10 μl) of resuspended cells were transferred to microscope slides, covered with coverslips, and sealed with nail polish. Slides were dried and stored at −20°C.Protein-mCherry fusions were examined using a Zeiss LSM 710 laser scanning microscope with a Plan-Apochromat 63×/1.40 oil lens. mCherry fluorescence was measured as excitation/emission at 561/578 to 696 nm. Images were recorded and analyzed using ZEN 2012 (Zeiss) and Image-Pro Plus 6.0 software .E. coli MC4100 relA+ strain were grown overnight in 10 ml of M9 medium with 100 μg/ml ampicillin at 37°C, 220 rpm. A 200-μl aliquot of overnight culture was subcultured (1:20 dilution) in 10 ml of LB in the presence of 100 μg/ml ampicillin. The cells were grown at 37°C and 220 rpm to an OD600 of 0.4. Protein-mCherry fusions were expressed from pBAD18-mcherry-derived plasmids induced by 10 mM arabinose. MazF was expressed from PSA1 plasmid induced by 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG). After 4 h of incubation at 37°C the samples were centrifuged at 14,000 rpm for 2 min. The supernatants were removed, and the pellets were resuspended in 0.5 ml of prewarmed M9 medium. The samples were serially diluted in prewarmed M9, plated on prewarmed LB plates, and incubated at 37°C. The percent survival was determined by dividing the number of colonies obtained from the “experiment” sample by the number of colonies obtained from the MC4100 relA+ sample used as the control.Single colonies of the 10.1128/mBio.01417-21.1Text S1Text S1, DOCX file, 0.01 MB.Primers used in this work. Download Copyright © 2021 Mager et al.2021Mager et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01417-21.7TABLE S1Table S1, DOCX file, 0.01 MB.Plasmids used in this work. Download Copyright © 2021 Mager et al.2021Mager et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the


The seafloor is considered the major sink for plastic debris in the world’s oceans. Biodegradable polymers are available on the market as a substitute for conventional plastic and could potentially end up in the same environment. To gain more insight into the effects of different sediments on the degradation rate of biodegradable plastic we performed two iterative seawater tank experiments. First, to test the effect of sediment grain size, film of Mater-Bi HF03V, a blend of thermoplastic starch and biodegradable polyesters, was placed on the surface of mud as well as on four different grain size fractions of beach sand. Disintegration half-life was shortest on mud (139 days) and increased with the grain size of the beach sediment fractions . We assume that the higher surface-to-volume ratio in fine sediment compared to coarse sediment led to a higher bacterial abundance and thus to faster disintegration rates. In a follow-up experiment, the <500 µm fraction of sediment from four different beaches around Isola d’Elba, Italy, was used to test plastic disintegration as above. Additionally, polyhydroxybutyrate  was used as a positive control and high-density polyethylene (HD-PE) as a negative control. No disintegration was observed for HD-PE. Mater-Bi HF03V and PHB disintegrated significantly differently on sediment from different sites, with half-lives of Mater-Bi HF03V ranging from 72 to 368 days and of PHB from 112 to 215 days. Here, the half-life was shortest on slightly coarser sediment and at potentially anthropogenically impacted sites. We assume that the effect of the grain size on the disintegration rate was masked by other parameters influencing the microbial community and activity. Understanding the parameters driving biodegradation is key to reliably report the range of disintegration rates occurring under the various conditions in different ecosystems. Plastic pollution occurs in all ecosystems on the planet, including deep-sea trenches , remote i.e. polymers and blends that degrade predominantly by the enzymatic action of microorganisms into CO2 , water, inorganic compounds, and biomass (e.g. geotextiles), the potential of being lost is high , or the input into the environment is unavoidable . As part of an assessment of potential environmental benefits and risks, the biodegradability of a material has to be proven before raising any claim thereof  are achieved by exposing the plastic samples in-situ or in mesocosms (tanks) with a much higher volume than the laboratory flasks . Also materials with a lower specific density or items containing lighter compartments such as bubbles or foam will eventually sink due to fouling  laying on the sediment surface was measured. In a follow-up experiment, sediment was collected from four different locations and directly put into aquaria. Here, plastic disintegration was tested the same way as above. Additionally, polyhydroxybutyrate (PHB) was used as a positive control and high-density polyethylene (HD-PE) as a negative control.In both experiments, the degradation of Mater-Bi HF03V, a blend of thermoplastic starch and biodegradable polyesters, was investigated . In the In the second experiment, two additional materials were tested as controls . PolyhydTwo experiments were performed in aquaria: to study the effect of sediment grain size on material disintegration, the disintegration of the biodegradable plastic film laying on different grain size fractions of beach sand, and additionally on mud, was tested in a pilot experiment. In a follow-up experiment, three plastic materials were tested on natural sediments from four different beaches.−1 . Aquaria were kept in a climate chamber for ten months at 21 °C  to prevent the potential loss of fragments and placed on the sediment surface of each test tank . After 5At four beaches around Isola d’Elba , sedimenn = 3 for each sediment treatment. All 12 tanks were sealed with a lid made of plastic film with a small hole to allow the passage of an air hose (Three glass aquaria (30 × 20 × 20 cm) were filled with a layer of 4 cm (2.4 L) of each of the four sediment types and 6 L of natural seawater collected in Seccheto , which lair hose .The salinity was regularly checked as described above and if necessary adjusted to 39 with distilled water. Dissolved oxygen was around 95% air saturation and pH around 8.1 . In contTest materials were cut to strips of 6 × 8 cm. Each specimen was put between two layers of polyester mesh  with a square-shaped mesh of 2 × 2 mm and held together at the short sides with plastic clamps. One replicate of each material was randomly placed in each aquarium.After the start of the experiment, sampling occurred after 129, 186 and 252 days for the experiments with Marina di Campo, Portoferraio, and Naregno sediment. The last sampling for the test with Fetovaia sediment was after 356 days because after 252 days, the disintegrated area of Mater-Bi HF03V in the Fetovaia treatment was estimated to be below 50% and much lower than in the other treatments. During sampling, one replicate of each material was retrieved from each of the three aquaria containing the same type of sediment. The upper mesh was carefully removed. Depending on the state of disintegration, the samples were photographed with or without the lower mesh. No re-exposure of the specimens occurred.Disintegration was determined photogrammetrically as described before . The rembetareg package) and the appropriate link-function  selected by comparing the Root Mean Square Deviation (package caret). Half-life was calculated by back-transforming link-functions for 50% material disintegration and solving the formula for the time (t0.5) using the coefficients estimated by the linear model. Monte-Carlo simulations were used to calculate 500,0000 half-live values considering the variance of the model coefficients, which allowed statistical tests with empirical p-values.All data was analysed in R  as descrThe test material Mater-Bi HF03V showed substantial disintegration on mud after 153 days and on all grain size fractions within 298 days .The half-life differed significantly between different sediment size fractions and was lowest for samples on mud (139 days), followed by samples on the sediment with a size fraction of 63–250 µm (296 days), 250–500 µm (310 days), and sediment above 500 µm  and SI-2Disintegration was obvious for the biodegradable materials Mater-Bi HF03V and PHB . HD-PE dThe predicted half-life for Mater-Bi HF03V ranged from 72 to 368 days and for PHB from 112 to 259 days . PHB dis−1 dry weight) than the other sediments  and negative control (HD-PE) was tested.Overall, the half-life of PHB (112 to 259 days) was in a similar range as in a previous study with similar settings. The disintegration of a not further specified type of Mater-Bi was tested in a benthic field experiment off Isola d’Elba at a depth of 36 m . Disintee.g. in municipal waste waters from Marina di Campo and Portoferraio may be more prepared or pre-adapted for the degradation of a more complex blend than the community on beaches low in anthropogenic organics input. A more in-depth study, comparing the composition and metabolic functionality of each of the beach sediments could help to explain the differences in the biologically driven disintegration of biodegradable plastic materials observed in this study.PHB disintegrated significantly faster on Marina di Campo sediment than on the other sediment types. Also, Mater-Bi HF03V disintegrated fastest on Marina di Campo sediment, but similarly fast on Portoferraio sediment. Different than expected from the results of the pilot study, the disintegration was faster for the coarser Marina di Campo sediment. The grain size characteristics of the Portoferraio sediment was not substantially different from those of Naregno and Fetovaia sediment  to SI-6.The disintegration of Mater-Bi HF03V on Fetovaia sediment and PHA on Nargeno sediment was highly variable. This could be caused by processes occurring during material disintegration. The increasing biodegradation weakens the structure of the film until reaching an unstable equilibrium. Then, even small differences in biodegradation rates have strong effects on the physical integrity of the sample and therefore its disintegration.From the pilot experiment, we conclude that in general there is a strong impact of sediment grain size on the disintegration rate. In natural beach sediment containing a mix of different grain sizes, no such clear effects could be observed and differences in material disintegration could be masked by additional environmental parameters. The impact of anthropogenically influenced sediment on the biodegradation rates of biodegradable polymers should be further investigated.10.7717/peerj.11981/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.11981/supp-2Supplemental Information 2were placed between two layers of mesh and directly exposed at the sediment surface. Each mesh was weighted with 4 pieces of granite to secure it on the sediment surface.Overview and individual tanks with sand of the grain size fractions of >1,000 µm, 500–1,000 µm, 250–500 µm, 63–250 µm, and mud. Sample strips of Mater-Bi HF03V Click here for additional data file.10.7717/peerj.11981/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.11981/supp-4Supplemental Information 4The sample film was protected by two layers of mesh and placed on the surface of sediment collected at different beaches. The lid covering the tank was weighted down with a granite tile. A hose connected to an air pump kept the oxygen concentration in the seawater at air-saturation. The tanks were placed in plastic bins.Click here for additional data file.10.7717/peerj.11981/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.11981/supp-6Supplemental Information 6During the first phase of the experiment, the tanks containing Marina di Campo and Portoferraio sediment were monitored, the same temperatures are assumed for the tanks containing Fetovaia and Naregno sediment. Tanks containing Fetovaia sediment were monitored in the last phase of the experiments after the other tests were terminated.Click here for additional data file.10.7717/peerj.11981/supp-7Supplemental Information 7Depending on the state of material disintegration, the sample films were either photographed without the lower mesh or on the lower mesh.Click here for additional data file.10.7717/peerj.11981/supp-8Supplemental Information 8CL = 95% confidence level.Click here for additional data file.10.7717/peerj.11981/supp-9Supplemental Information 9p-values in bold.Significant Click here for additional data file.10.7717/peerj.11981/supp-10Supplemental Information 10i.e. below detection limit.One sample of bulk seawater before the experiments was analysed. TC, total carbon; TOC, total organic carbon; DOC, dissolved organic carbon; TIC, total inorganic carbon; POC, particulate organic carbon; DOP, dissolver organic carbon; TIN, total inorganic nitrogen; TON, total organic nitrogen; n.d., not detectable, Click here for additional data file.10.7717/peerj.11981/supp-11Supplemental Information 11CL = 95% confidence levelClick here for additional data file.10.7717/peerj.11981/supp-12Supplemental Information 12p-values in bold.Significant Click here for additional data file.10.7717/peerj.11981/supp-13Supplemental Information 13p-values in bold.Significant Click here for additional data file.10.7717/peerj.11981/supp-14Supplemental Information 14rep, replicate number; sed, sediment type/grain size fraction; perc_degr, disintegrated area in %; days, exposed time in days.Click here for additional data file.10.7717/peerj.11981/supp-15Supplemental Information 15rep, replicate number; material, polymer material; beach, beach where sediment was collected; perc_degr, disintegrated area in %; days, exposed time in days.Click here for additional data file.


Background: Accumulation of inflammatory leukocytes in articular tissues is the hallmark feature of rheumatoid arthritis (RA). Increasing evidence from observational studies has suggested that several cytokines may be involved in the development of RA. However, traditional observational studies are susceptible to bias from confounding and reverse causation; therefore, the potential causal relationships of individual cytokines with the risk of RA remain elusive.Objective: In this study, we evaluated whether genetically determined circulating levels of cytokines were associated with the risk of RA by performing Mendelian randomization (MR).Methods: We identified single nucleotide polymorphisms (SNPs) associated with circulating levels of cytokines and growth factors from a genome-wide association study (GWAS) including 8,293 participants of Finnish ancestry as instrumental variables (IVs). The association estimates of these IVs with the risk of RA were obtained from a GWAS meta-analysis including 14,361 RA cases and 43,923 controls of European ancestry. We conducted a series of MR analyses to assess the relationship between genetically determined circulating cytokines and the risk of RA, including the random-effects inverse variance-weighted, weighted-median, MR-Egger regression, and MR pleiotropy residual sum and outlier tests. For potential cytokine-RA associations supported by MR evidence, sensitivity analyses were further performed using restricted IV sets of SNPs with colocalization evidence and that excluding pleiotropic SNPs.Results: In the primary MR analysis, there was a suggestive inverse association between genetically determined circulating level of macrophage inflammatory protein-1β (MIP-1b) and the risk of RA . The effect estimates were similar in alternative MR analyses. Among SNPs used as IVs for MIP-1b, we found 92 SNPs without documented pleiotropy and three SNPs with evidence of colocalization. The association of MIP-1b with RA from sensitivity analyses using these two sets of restricted IVs remained stable.Conclusion: Our study suggests that genetically determined elevated circulating level of MIP-1b may be associated with a lower risk of RA. Further studies are warranted to determine how MIP-1b and related pathways may contribute to the development of RA. Lymphoc T cells . Improve T cells . However T cells , and up  T cells . AlternaAccumulation of inflammatory leukocytes in articular tissues is the hallmark feature of RA. Why articular tissues are preferentially affected, and the process of cellular recruitment into the inflamed tissue, remains unclear. Increasing evidence from observational studies has suggested that cytokines, such as monocyte chemoattractant protein-1 and interleukin-1 α, might be involved in the disease process . HoweverMendelian randomization (MR) is a method that uses genetic variants as instrumental variables (IVs) for the exposures and assesses their associations with disease outcomes . Unlike In this study, we systematically evaluated the relationships between circulating levels of cytokines and the risk of RA by conducting a two-sample MR study in a large GWAS meta-analysis of 14,361 participants with RA and 43,923 control participants . SensitiThe overall design of the study is summarized in p-value for Hardy–Weinberg equilibrium ≤1.0 × 10–6 in all samples were also excluded  for their associations with circulating levels of cytokines. Then, for each cytokine, SNPs used as IVs were pruned for LD at a stringent threshold (r2 < 0.1), and those with a larger p-value for the exposure of interest were excluded. We identified a total of 958 SNPs associated with circulating levels of 28 cytokines. We further excluded SNPs for which associations of these SNPs and their proxies (SNPs correlated at r2 > 0.9) were not available in RA summary statistics, leaving a total of 270 SNPs (including one proxy) as the final IV sets for the 27 circulating cytokines (We selected SNPs reaching the genome-wide level of significance (ytokines .p < 5 × 10−8 with other traits using the Open Targets (Last accessed on 9 October 2021). Additionally, a restricted IV set were constructed including independent SNPs with colocalization evidence.In sensitivity analyses, we used several alternative sets of IVs. Specifically, to minimize the effect of potential pleiotropic SNPs on causal association estimates, we excluded pleiotropic SNPs showing associations of R2)   . F-statiescribed . We thenescribed . We perfescribed  as primaescribed . Additioiotropy) . The posiotropy) .We further included SNPs within ±1 megabase region of the SNPs associated with circulating levels of cytokines at genome-wide significance level to form candidate SNP sets. Then, we extracted the association estimates of these SNPs with the risk of RA. Based on the assumption that there was one genetic variant within each region shared across two phenotypes , the Bayesian framework was used to test the posterior probability of association (PPA) for four hypotheses, according to default colocalization protocols . For SNPp-value < 0.002 (0.05/27) based on the number of cytokines included in the primary MR analyses. p-value above 0.002 but less than 0.05 were considered suggestive evidence for an observed association. All analyses were performed in R software (v 3.6.2) using coloc . Notably, circulating levels of macrophage inflammatory protein-1β (MIP-1b/CCL4) showed suggestive inverse association with the risk of RA  using the random-effects IVW method (The primary MR results from the random-effects IVW method are presented in W method . The detp = 0.034). Moreover, there was no evidence showing the presence of directional pleiotropy effects as assessed by MR-Egger regression (P for intercept = 0.859), and no outliers were detected using the MR-PRESSO test .In sensitivity analyses using alternative IV sets, we first systemically scanned individual SNPs used as IVs for MIP-1b for potential pleiotropy. We found that no SNPs were associated with any other cytokines at a genome-wide significance level, but there were two pleiotropic variants, based on the Open Targets website . After r2 < 0.1), three independent non-pleiotropic SNPs remained in the final IV set. MR analyses based on this restricted IV set yielded a similar association between circulating MIP-1b and the risk of RA .Furthermore, based on the observed MR evidence, we conducted colocalization analysis of RA and MIP-1b. We found a total of 10 SNPs with colocalization evidence for circulating MIP-1b and RA . After pBy using two-sample MR and colocalization, we systematically assessed the potential causal relationships between circulating levels of cytokines and the risk of RA. Our study found a suggestive inverse association between genetically determined circulating level of MIP-1b and the risk of RA. Moreover, the results for the inverse association were stable using alternative MR methods and in sensitivity analyses using alternative IV sets. Collectively, these findings provided evidence for a potential protective role of circulating MIP-1b in the development of RA.p < 0.01) (p = 0.007)  . In anot < 0.01) , serum M= 0.007) , which a= 0.007) . Differe= 0.007) .One fundamental assumption for MR study to ensure a valid causal estimate is that genetic variants used as IVs did not directly act on the target outcome, neither affect the outcome through pathways other than through the exposure . To veriThe underlying biological mechanism of MIP-1b in the pathogenesis of RA remains unclear. One possible explanation has been suggested is that MIP-1b is involved in the regulation of Th1 cell trafficking . EvidencCCL4 gene polymorphisms) on normal development may influence the expression of other genes, which in turn compensate for the influence of lifelong higher circulating level of MIP-1b (There were several limitations to this study. First, our results may not be generalizable to other populations, since the ancestry of participants included in our MR study was restricted to Europeans. Second, the effects of genetic variations (e.g. elevated MIP-1b level due to f MIP-1b . Hence, Our MR study found suggestive evidence that genetically determined elevated circulating level of MIP-1b was associated with a reduced risk of RA. More studies on the potential biological mechanism underlying MIP-1b and risk of RA are warranted to explore whether MIP-1b or related pathways could be potential therapeutic targets in the prevention of RA.